Hemodynamic Changes in the Masseter and Superior Orbicularis Oris Muscles before and after Exercise Load: A Comparison between Young Adult Women and Middle-Aged to Old Adult Women.

Background: The vascularity index (VI) is useful for measuring the hemodynamics on ultrasound imaging. However, there are no reports concerning the application of the VI to facial muscles. Objective: The aim of this study was (1) to establish a method of measuring the hemodynamics in facial muscles in a constant way and (2) to evaluate the hemodynamic changes in the masseter and superior orbicularis oris muscles (SOOMs) before and after exercise load in two subject groups of females of different ages. Methods: (1) The VI in the SOOM was calculated, and the test-retest reliability was assessed in seven healthy adults. (2) The VIs in the left-side masseter and SOOM were calculated in 3 sessions: before exercise loading (T0), immediately after loading (T1), and 5 minutes after T1 (T2) for the young adult group (YAG, n = 20; age range, 20-35 years) and the middle-aged to old group (MOG, n = 20; age range, 50-70 years). Tasks were gum chewing for the masseter muscle and lip sealing for the SOOM. The differences in the mean peak flows between two sessions were examined. Results: (1) Significant differences were not noted for the repeatedly measured average volumes of blood flow with good test-retest agreement (intraclass correlation coefficient = 0.81). (2) In both muscles of the YAG, there were a significant increase in T1 compared with T0 and a significant decrease in T2 compared with T1 (all p < 0.05). In both muscles of the MOG, no significant differences were noted in either comparison. Conclusions: A method of measuring the hemodynamics in facial muscles was developed and showed good reliability. Changes in the blood flow after exercise load in these muscles may vary with age in women.

Low-carbohydrate diet by staple change attenuates postprandial GIP and CPR levels in type 2 diabetes patients.

AIMS: The aim of this study is to investigate the effects of a low-carbohydrate staple food (i.e., low-carbohydrate bread) on glucose and lipid metabolism and pancreatic and enteroendocrine hormone secretion in comparison with meals containing normal-carbohydrate bread, without consideration of the carbohydrate content of the side dishes. METHODS: T2DM patients (n = 41) were provided meals containing low-carbohydrate bread (LB) together with side dishes or normal-carbohydrate bread (NB) together with side dishes every other day as a breakfast. Blood glucose levels were evaluated by using a continuous glucose monitoring system; blood samples were collected before and 1 and 2 h after the breakfast. RESULTS: Postprandial blood glucose levels, plasma insulin, plasma glucose-dependent insulinotropic polypeptide (GIP) and plasma triglyceride were significantly lower and plasma glucagon levels were significantly higher in LB compared with those in NB. Plasma glucagon-like peptide-1 (GLP-1) levels did not differ in the LB and NB groups. CONCLUSIONS: These results indicate that changing only the carbohydrate content of the staple food has benefits on glucose and lipid metabolism in T2DM patients concomitant with the decrease of insulin and GIP secretion, which ameliorate body weight gain and insulin resistance.

Intestinal TGR5 agonism improves hepatic steatosis and insulin sensitivity in Western diet-fed mice.

Takeda G protein-coupled receptor 5 (TGR5) agonists induce systemic release of glucagon-like peptides (GLPs) from intestinal L cells, a potentially therapeutic action against metabolic diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and Type 2 diabetes. Historically, TGR5 agonist use has been hindered by side effects, including inhibition of gallbladder emptying. Here, we characterize RDX8940, a novel, orally administered TGR5 agonist designed to have minimal systemic effects and investigate its activity in mice fed a Western diet, a model of NAFLD and mild insulin resistance. Agonist activity, binding selectivity, toxicity, solubility, and permeability of RDX8940 were characterized in standard in vitro models. RDX8940 pharmacokinetics and effects on GLP secretion, insulin sensitivity, and liver steatosis were assessed in C57BL/6 mice fed normal or Western diet chow and given single or repeated doses of RDX8940 or vehicle, with or without dipeptidyl peptidase-4 (DPP4) inhibitors. Gallbladder effects were assessed in CD-1 mice fed normal chow and given RDX8940 or a systemic TGR5 agonist or vehicle. Our results showed that RDX8940 is minimally systemic, potent, and selective, and induces incretin (GLP-1, GLP-2, and peptide YY) secretion. RDX8940-induced increases in plasma active GLP-1 (aGLP-1) levels were enhanced by repeated dosing and by coadministration of DPP4 inhibitors. RDX8940 increased hepatic exposure to aGLP-1 without requiring coadministration of a DPP4 inhibitor. In mice fed a Western diet, RDX8940 improved liver steatosis and insulin sensitivity. Unlike systemic TGR5 agonists, RDX8940 did not inhibit gallbladder emptying. These results indicate that RDX8940 may have therapeutic potential in patients with NAFLD/NASH. NEW & NOTEWORTHY Takeda G protein-coupled receptor 5 (TGR5) agonists have potential as a treatment for nonalcoholic steatohepatitis and nonalcoholic fatty liver disease (NAFLD) but have until now been associated with undesirable side effects associated with systemic TGR5 agonism, including blockade of gallbladder emptying. We demonstrate that RDX8940, a potent, selective, minimally systemic oral TGR5 agonist, improves liver steatosis and insulin sensitivity in a mouse model of NAFLD and does not inhibit gallbladder emptying in mice.

Synchronization in flickering of three-coupled candle flames.

When two or more candle flames are fused by approaching them together, the resulting large flame often exhibits flickering, i.e., prolonged high-frequency oscillation in its size and luminance. In the present work, we investigate the collective behaviour of three-coupled candle flame oscillators in a triangular arrangement. The system showed four distinct types of syncronised modes as a consequence of spontaneous symmetry breaking. The modes obtained include the in-phase mode, the partial in-phase mode, the rotation mode, and an anomalous one called the "death" mode that causes a sudden stop of the flame oscillation followed by self-sustained stable combustion. We also clarified the correlation between the inter-flame distance and the frequency with which the modes occur.

The Possible Role of TASK Channels in Rank-Ordered Recruitment of Motoneurons in the Dorsolateral Part of the Trigeminal Motor Nucleus.

Because a rank-ordered recruitment of motor units occurs during isometric contraction of jaw-closing muscles, jaw-closing motoneurons (MNs) may be recruited in a manner dependent on their soma sizes or input resistances (IRs). In the dorsolateral part of the trigeminal motor nucleus (dl-TMN) in rats, MNs abundantly express TWIK (two-pore domain weak inwardly rectifying K channel)-related acid-sensitive-K(+) channel (TASK)-1 and TASK3 channels, which determine the IR and resting membrane potential. Here we examined how TASK channels are involved in IR-dependent activation/recruitment of MNs in the rat dl-TMN by using multiple methods. The real-time PCR study revealed that single large MNs (>35 µm) expressed TASK1 and TASK3 mRNAs more abundantly compared with single small MNs (15-20 µm). The immunohistochemistry revealed that TASK1 and TASK3 channels were complementarily distributed in somata and dendrites of MNs, respectively. The density of TASK1 channels seemed to increase with a decrease in soma diameter while there were inverse relationships between the soma size of MNs and IR, resting membrane potential, or spike threshold. Dual whole-cell recordings obtained from smaller and larger MNs revealed that the recruitment of MNs depends on their IRs in response to repetitive stimulation of the presumed Ia afferents. 8-Bromoguanosine-cGMP decreased IRs in small MNs, while it hardly changed those in large MNs, and subsequently decreased the difference in spike-onset latency between the smaller and larger MNs, causing a synchronous activation of MNs. These results suggest that TASK channels play critical roles in rank-ordered recruitment of MNs in the dl-TMN.

Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing.

We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.

Labeling of islet cells with iron oxide nanoparticles through DNA hybridization for highly sensitive detection by MRI.

A labeling method for islet cells with superparamagnetic iron oxide nanoparticles (SPIOs) based on DNA hybridization is proposed for monitoring of transplanted islets by magnetic resonance imaging (MRI). The surfaces of SPIOs were modified by via Michael reaction by reacting oligo-(deoxyadenylic acid)-bearing a terminal thiol group at the 5'-end ((dA)20-SH) with maleic acid functional groups on the SPIOs. The SPIOs were immobilized on islet cells which had been pretreated with oligo-(thymidylic acid)-poly(ethylene glycol)-phospholipid conjugates ((dT)20-PEG-DPPE) through DNA hybridization. Transmission electron microscopy observations revealed that SPIOs were initially anchored on the islet cell surfaces and subsequently transferred to endosomes or exfoliated with time. The SPIO-labeled islet cells could be clearly detected as dark spots by T2(*)-weighted MR image, whereas non-labeled islet cells could not be detected.

The cellular expression of GABA(A) receptor alpha1 subunit during spermatogenesis in the mouse testis.

GABA(A) receptors are pentamers in structure and are mainly composed of alpha, beta and gamma subunits. These receptors are known to function as chloride channels. We observed alpha5, beta1 and gamma3 subunit immunoreactivity in the mouse testes, specifically in the cytoplasm surrounding the nucleus in the spermatocytes and spermatids. In the current study, alpha1 subunit immunoreactivity was located in the nucleus of spermatogonia, spermatocytes and round spermatids. Immunoelectron microscopy revealed that the alpha1 subunit was localized within the nucleus of pachytene and diplotene spermatocytes in the area of condensed chromatin rather than extended chromatin. Protein sequence analysis revealed that the alpha1 subunit included DM DNA binding domains that were related to transcription factors involved in testicular differentiation in adult mice. These findings suggest that the alpha1 subunit may undertake a gene transcription function during the maturation of germ cells. a1 immunoreactivity was also detected within the mitochondria of spermatocytes and in the acrosome of round and elongated spermatids. Although the precise physiological role of the GABA(A) receptor alpha1 subunit in mitochondria remains unknown, we hypothesize that its function in the acrosome may be related to the acrosome reaction during fertilization or during spermatogenesis.

A novel glycerophosphodiester phosphodiesterase, GDE5, controls skeletal muscle development via a non-enzymatic mechanism.

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5DeltaC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5DeltaC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.

Possibility of using nerve segments dissected from human cadavers for grafting: preliminary report.

An intercostal nerve obtained from a human cadaver 6 h post-mortem was transplanted into the rat sciatic nerve and nerve regeneration was observed 4 and 8 weeks after surgery. Sciatic nerves from deceased rats up to 2 days post-mortem were also transplanted for comparison. Good nerve regeneration was observed through the human cadaver-derived graft to the distal segment at the medial plantal nerve 8 weeks after surgery. The results of the present study indicate the possibility that nerves from human cadavers can be used for nerve grafting in clinical applications.

Cellular localization of GABA and GABAB receptor subunit proteins during spermiogenesis in rat testis.

The GABAergic system, a major inhibitory regulator in the central nervous system, may also play important roles in peripheral nonneuronal tissues and cells. Recent studies showed that GABAB receptor is expressed in testis and sperm. To understand the role of the GABAergic system in spermiogenesis, we examined cellular localization of GABA and GABAB receptor subunits in rat spermatids by immunocytochemistry. Immunoreactivity for GABA was detected around acrosomal granules of spermatids during the Golgi and cap phases. GABAB1 immunoreactivity was observed in the acrosomal vesicle of spermatids in Golgi phase, and during cap phase, this reactivity expanded to the entire region of the acrosome covering the nuclear membrane. The level of reactivity decreased gradually with maturation of spermatids. In contrast, GABAB2 immunoreactivity was not observed in spermatids during Golgi phase but was detected in the equatorial region during cap phase. Both GABA immunoreactivity and GABAB2 immunoreactivity were transferred to the residual cytoplasm during the release of spermatozoa. Electron microscopic immunocytochemistry revealed that, during cap phase, GABA and GABAB1 were distributed within the whole acrosomal vesicle but not in the acrosomal granule. GABAB2 immunoreactivity was observed in the narrow space between the inner acrosomal and nuclear membrane and was limited to the equatorial region of the spermatid head. These results indicate that the GABAergic system might be involved in regulation of spermiogenesis.

Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex.

Gamma-aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.

Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions. An immunohistochemical tract-tracing study in the GAD67-GFP knock-in mouse.

It has been reported in the cat and rat that inhibitory premotor neurons, which send their axons to motoneurons of the trigeminal motor nucleus (Vm) are distributed in the reticular regions around the Vm, especially in the supratrigeminal region (Vsup) and the intertrigeminal region (Vint). In the present study, we examined neuronal connections of GABAergic neurons in the Vsup and Vint in the mouse by utilizing the adult heterozygous GAD67-GFP knock-in mouse, in which green fluorescence protein (GFP) is expressed in GABAergic neurons under the control of the endogenous GAD (GAD67) gene promoter [Yanagawa, Y., Kaneko, K., Kanbara, N., Totsuka, M., Yagi, T., Obata, K., 2001. Development of mouse expressing GFP in GABAergic neurons. Neurosci. Res. Suppl. 25, S77; Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J.-I., Obata, K., Kaneko, T., 2003. Green fluorescent protein expression and colocalization with calretinin, parvalbumin and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60-79]. The connections were examined light- and electron-microscopically by combining the anterograde or the retrograde tract-tracing method with the immunohistochemical method for GFP. The data indicated that the Vsup and Vint of the mouse contained GABAergic neurons, which received projection fibers from the marginal layer of the nucleus of the spinal tract of the trigeminal nerve (Vc) on the ipsilateral side and sent their axons to the Vm on the contralateral side. Some of these GABAergic neurons may represent Vm-premotor neurons that receive nociceptive input from the Vc to elicit jaw-opening reflex by inhibiting jaw-closing Vm-motoneurons.

Presynaptic localization of an AMPA-type glutamate receptor in corticostriatal and thalamostriatal axon terminals.

The neostriatum is known to receive glutamatergic projections from the cerebral cortex and thalamic nuclei. Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) are located on axon terminals of corticostriatal and thalamostriatal afferents, respectively, whereas VGluT3 is found in axon terminals of cholinergic interneurons in the neostriatum. In the present study, the postsynaptic localization of ionotropic glutamate receptors was examined in rat neostriatum by the postembedding immunogold method for double labelling of VGluT and glutamate receptors. Immunoreactive gold particles for AMPA receptor subunits GluR1 and GluR2/3 were frequently found not only on postsynaptic but also on presynaptic profiles immunopositive for VGluT1 and VGluT2 in the neostriatum, and GluR4-immunoreactive particles were observed on postsynaptic and presynaptic profiles positive for VGluT1. Quantitative analysis revealed that 27-45% of GluR1-, GluR2-, GluR2/3- and GluR4-immunopositive particles found in VGluT1- or VGluT2-positive synaptic structures in the neostriatum were associated with the presynaptic profiles of VGluT-positive axons. In contrast, VGluT-positive presynaptic profiles in the neostriatum showed almost no immunoreactivity for NMDA receptor subunits NR1 or NR2A/B. Furthermore, almost no GluR2/3-immunopositive particles were observed in presynaptic profiles of VGluT3-positive (cholinergic) terminals that made asymmetric synapses in the neostriatum, or in those of VGluT1- or VGluT2-positive terminals in the neocortex. The present results indicate that AMPA receptor subunits but not NMDA receptor subunits are located on axon terminals of corticostriatal and thalamostriatal afferents, and suggest that glutamate released from these axon terminals controls the activity of the terminals through the presynaptic AMPA autoreceptors.

Intracellularly labeled pyramidal neurons in the cortical areas projecting to the spinal cord. II. Intra- and juxta-columnar projection of pyramidal neurons to corticospinal neurons.

Intra- or juxta-columnar connections of pyramidal neurons to corticospinal neurons in rat motorsensory cortices were examined with brain slices by combining intracellular staining with Golgi-like retrograde labeling of corticospinal neurons. Of 108 intracellularly labeled pyramidal neurons, 27 neurons were selected for morphological analysis by successful staining of their axonal arborizations and sufficient retrograde labeling of corticospinal neurons. Many varicosities of local axon collaterals of each pyramidal neuron were closely apposed to the dendrites of corticospinal neurons, suggesting the convergent projections of layer II-VI pyramidal neurons to corticospinal neurons. Particularly, the varicosities of a layer IV star-pyramidal neuron made two- to three-fold more appositions to the dendrites of corticospinal neurons than those of a pyramidal neuron in the other layers. Fifteen appositions were examined electron-microscopically and 60% of them made asymmetric axospinous synapses. The present results together with those of the preceding report suggest that thalamic inputs are conveyed to corticospinal neurons preferentially via layer IV star-pyramidal neurons with phasic response properties, and thereby might contribute to the initiation or switching of movement. In contrast, inputs with tonic response properties from the other layers seem to be integrated in corticospinal neurons, and might be useful in maintaining the activity of corticospinal neurons.

Leaving group effects in gas-phase substitutions and eliminations.

Using a methodology recently developed for studying the product distributions of gas-phase S(N)2 and E2 reactions, the effect of the leaving group on the reaction rate and branching ratio was investigated. Using a dianion as the nucleophile, reactions with a series of alkyl bromides, iodides, and trifluoroacetates were examined. The alkyl groups in the study are ethyl, n-propyl, n-butyl, isobutyl, isopropyl, sec-butyl, and tert-butyl. The data indicate that leaving group abilities are directly related to the exothermicities of the reaction processes in both the gas phase and the condensed phase. Gas-phase data give a reactivity order of iodide > trifluoroacetate > bromide for S(N)2 and E2 reactions. Previous condensed phase data indicate a reactivity order of iodide > bromide > trifluoroacetate for substitution reactions; however, the basicities of bromide and trifluoroacetate are reversed in the condensed phase so this reactivity pattern does reflect the relative reaction exothermicities. Aside from this variation, the gas-phase data parallel condensed phase data indicating that the substituent effects are rooted in the nature of the alkyl substrate rather than in differences in solvation. The experimental data are supported by calculations at the MP2/6-311+G(d,p)//MP2/6-31+(d) level.

Stereoselectivity in the collision-activated reactions of gas phase salt complexes.

The collision-activated dissociations (CAD) of gas phase salt complexes composed of chiral ions were studied in a quadrupole ion trap mass spectrometer. Because both partners in the salt are chiral, diastereomeric complexes can be formed (e.g., RR, RS). Two general types of complexes were investigated. In the first, the complex was composed of deprotonated binaphthol and a chiral bis-tetraalkylammonium dication. CAD of these complexes leads to the transfer of a proton or an alkyl cation to the binaphtholate leading to a singly-charged tetraalkylammonium cation. During CAD, diastereomeric complexes give significantly different product distributions indicating reasonable stereoselectivity in the process. In the second system, the complexes involved a peptide dianion and a chiral tetraalkylammonium cation. These systems may be viewed as very simple models for the interactions of peptides/proteins with small chiral molecules. Again, stereoselectivity was evident during CAD, but the extent was dependent on the nature of the peptide and not observable in some cases. To better understand the structural features needed to achieve stereoselectivity in gas phase salt complexes, representative transition states were modeled computationally. The results suggest that it is critical for the asymmetry of the nucleophile (i.e., anion) to be well represented in the vicinity of its reactive center.

Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents.

Expression of vesicular glutamate transporters (VGLUTs: VGLUT1, VGLUT2 and VGLUT3) in muscle spindle afferents was examined in rats. VGLUT1 immunoreactivity was detected in the sensory endings on the equatorial and juxta-equatarial regions of intrafusal fibers as well as in many axon terminals within lamina IX of the spinal cord. VGLUT1 might be expressed not only in the central axon terminals but also in the peripheral sensory endings of muscle-spindle afferents.

Independent inputs by VGLUT2- and VGLUT3-positive glutamatergic terminals onto rat sympathetic preganglionic neurons.

To characterize glutamatergic axon terminals onto sympathetic preganglionic neurons (SPNs), we visualized immunohistochemically three vesicular glutamate transporters (VGLUTs) in the intermediolateral cell column (IML) of rat thoracic spinal cord. VGLUT2 and VGLUT3 immunoreactivities but not VGLUT1 immunoreactivity were distributed in the IML and found in terminals making asymmetric synapses and apposed to dendrites immunopositive for choline acetyltransferase, an SPN marker. VGLUT2 and VGLUT3 immunoreactivities were not co-localized with each other. A population of VGLUT2-immunoreactive but not VGLUT3-immunoreactive terminals were adrenergic or noradrenergic. Some of VGLUT3-immunoreactive but not VGLUT2-immunoreactive terminals contained serotonin. These results indicate at least two independent glutamatergic terminal populations, which include a distinct monoaminergic subpopulation, making excitatory inputs onto SPNs.

Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation.

To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1- and VGluT2-immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1-immunolabeled terminals were smaller than VGluT2-labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra- and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons.